The present invention relates to methods of predicting the outcome of infection. In particular it relates to methods for predicting the outcome of viral infections, including HBV infection, as well as methods for determining the susceptibility of children to infection. The invention also includes kits for use in such methods.
Mannose binding protein (MBP) is a calcium dependent lectin that plays an important role in innate immunity both by activating complement and phagocytosis, using an MBP associated serine protease (MASP), and acting directly as an opsonin by binding to collectin receptors through the collagen domain of MB?(Taylor et al, Biochem. J. 262:763-771 (1989); Kuhlman et al, J. Exp. Med., 169:1733-1745 (1989); Ohta et al, J. Biol. Chem., 265:1980-1984 (1990); Matsushita, M. and Fujita, T., J. Exp. Med., 176:1497-1502 (1992); Super et al, Clin. Exp. Immunol., 79:144-150 (1990); Malhotra et al, Biochem. J., 293:15-19 (1993)). MBP binds to carbohydrate moieties on various pathogens including influenza A virus (Hartshorn et al, J. Clin. Invest., 91:1414-1420 (1993)). Point mutations in codons 54 and 57 of the MBP gene are associated with low serum levels of MBP and a common immunodeficiency caused by an opsonic defect (Sumiya et al, Lancet, 337:1569-1570 (1992); Lipscombe et al, Human Mol. Genet., 1:709-715 (1992)). A less common mutation in codon 52 has also been reported in associated with low serum MBP levels (Madsen et al, Immunogenetics, 40:37-44 (1994)). Serum MBP levels may also be modulated by promoter polymorphisms (Madsen et al, J. Immunology, 3013-3020 (1995)). The mutations have been associated with severe and unusual infections in children and adults (Sumiya et al, (1992), supra; Summerfield et al, Lancet, 345:886-889 (1995); Garred et al, Lancet, 346:941-943 (1995)).
WO-A-8901519 discloses the sequence of human mannose binding protein, and its cloning. WO-A-9207579 discloses recombinantly produced portions of mannose binding proteins, including human mannose binding protein.
We have now shown a clear linkage between mutations in the gene coding for MBP and a subject""s susceptibility to infection.
Thus, in a first aspect the present invention provides a method of predicting and/or assessing the susceptibility of a subject to infection which comprises the step of determining whether the subject carries one or more mutations of the MBP gene.
Chronic fatigue syndrome is a condition of unknown aetiology, characterised by physical and mental fatigue, with other symptoms including myalgia. The symptoms typically follow a viral infection, which is often glandular fever or influenza-like. It has been suggested that persistent virus infections with EBV (Tobi, et al, Lancet, :61-4 (1982)), HHV6 or enteroviral infection (Behan, et al, J. Infect, 10:211-222 (1985)) could account for the syndrome, but others have suggested that the subjective nature of the symptoms, and occurrence of other symptoms such as altered sleeping pattern, are more typical of depressive disease or somatisation disorder (Manu, et al, Int. J. Psychiatry Med., 22:397-408 (1992)).
We have now shown that development of chronic fatigue syndrome is linked with the same mutation in the MBP gene. This predisposition seems to be linked to a susceptibility to chronic viral infection generally.
Thus, in a second aspect, the present invention provides a method of predicting and/or assessing the susceptibility of a subject to develop a chronic viral infection which comprises the step of determining whether the subject carries the codon 52 mutation of the MBP gene.
In a third aspect, the present invention provides a method of predicting and/or assessing the susceptibility of a subject to develop chronic fatigue syndrome which comprises the step of determining whether the subject carries the codon 52 mutation of the MBP gene.
In a fourth aspect, the present invention provides a method of diagnosing chronic fatigue syndrome in a subject which comprises the step of determining whether the subject carries the codon 52 mutation of the MBP gene.
Thus, there are provided, for the first time, clear methods for assessing patients with respect to their susceptibity to viral infections and subsequent development of chronic fatigue syndrome, as well as methods for diagnosing chronic fatigue syndrome in patients presenting with the symptoms thereof.
In addition, it will be possible to assess patients who have already contracted a viral infection, and this may lead to the ability to preempt development of the syndrome by treatment with non-mutant mannose binding protein.
In addition, there are clear similarities and overlap in the symptom complex of chronic fatigue syndrome and other conditions such as depressive disease, irritable bowel syndrome ad Gulf war syndrome. Therefore, it is believed that identification of the codon 52 mutation will also indicate a susceptibility to develop such conditions.
Thus, in a fifth aspect, the present invention provides a method of predicting and/or assessing the susceptibility of a subject to develop depressive disease, irritable bowel syndrome and/or gulf war syndrome, which comprises the step of determining whether the subject carries the codon 52 mutation of the MBP gene.
Exposure to hepatitis B virus (HBV) causes either no infection, fulminant hepatitis, a self limiting acute hepatitis or a chronic persistent infection that may progress to cirrhosis and/or hepatocellular carcinoma. The reasons for this variation in natural history of HBV are unknown but are probably determined by host immune factors.
The middle surface protein of the HBV envelope contains a mannose rich oligosaccharide that would bind to MBP (Gerlich, W. Structure and Molecular Biology; In viral Hepatitis, ed. Zuckerman A. J., Thomas H. C.; Churchill Livingstone page 93 (1993)) and previous studies in patients with chronic HBV have shown a serum opsonic defect (Munoz L., PhD thesis, (1989) University of London, page 89).
Clearly, it would be advantageous for a physician to be able to predict the whether a particular subject is likely to develop a chronic HBV infection.
We have now shown that development of chronic HBV infection is linked with mutations in MBP.
Thus, in a sixth aspect, the present invention provides a method of predicting the outcome of HBV infection in a subject which comprises the step of determining whether the subject carries a mutation in the MBP gene.
In a seventh aspect, the invention provides a method of predicting the susceptibility of a subject to development of chronic HBV infection which comprises the step of determining whether the subject carries a mutation in the MBP gene.
In an eighth aspect, the present invention provides a method of predicting the susceptibility of a subject to HBV infection and/or development of acute HBV infection which comprises the step of determining whether the subject carries a mutation in the MBP gene.
In particular, these methods of the invention will comprise a determination of the presence of a mutation at codon 52 of exon 1 of the MBP gene.
The ability to determine whether a patient is likely to progress from acute to chronic HBV infection is of great use to the physician. Such patients can be treated earlier, before chronic HBV infection develops. This, in turn, should lead to better rates of response. Patients with acute HBV infection can simply be screened, and those carrying the mutation can be targeted for treatment.
In addition, individuals can be identified who are susceptible to HBV infection and are therefore in particular need of vaccination. Those identified as carrying the mutation can thus be the subject of targeted vaccination programmes.
Repeated bacterial and fungal infections associated with MBP mutations have been reported in children and adults suspected of having a ummunodeficiency syndrome (Sumiya et al, (1991), supra; Summerfield et al, Lancet, 345:886-889; Garred et al, Lancet, 346:941-943 (1995)). Both MBP mutations and childhood infections are common but as yet no clear link between the presence of such mutations and a predisposition to childhood infectins has been demonstrated.
We have now shown that such a link exists, and that testing for the presence of mutations in the mannose binding protein enables identification of those children more at risk of infectious disease, and particularly those children who possess a predisposition to recurrent infection.
Thus, in a ninth aspect, the present invention provides a method of determining whether a child is more likely to be susceptible to infection which comprises the step of determining whether the child carries at least one mutation in the MBP gene.
In a tenth aspect, the invention provides a method of determining whether a child is more likely to be susceptible to recurrent infection which comprises the step of determining whether the child carries at least one mutation the MBP gene.
In an eleventh aspect, the present invention provides a method of determining whether a baby is more likely to be born prematurely which comprises the step of determining whether the baby carries at least one mutation in the MBP gene.
In a twelfth aspect, the present invention provides a method of determining whether a child is more likely to be susceptible to infection which comprises the step of determining whether the child is homozygous for an MBP mutation. In the context of this aspect of the invention, homozygous refers either to the genotype or phenotype of the child with respect to MBP mutation. In other words, a child will be regarded as homozygous for MBP mutation even if it possesses a different mutation in each copy of the MBP gene.
In particular, these methods of the invention will comprise a determination of the presence of a mutation at codons 52, 54 or 57 of exon 1 of the MBP gene.
Suitably, in the methods of the present invention, the subject""s genomic DNA will be extracted from a suitable source, e.g. from a blood sample, and the exon 1 region of the MBP gene will be amplified prior to analysis. Amplification can be carried out using various methods known to the skilled man, and include PCR, a technique well known to the skilled man, Ligase Chain Reaction (Abravaya et al, Nucleic Acid Res. 23: 675-82 (1995)), amplification refractory mutation system PCR (Davies et al, Arthritis and Rheumatism, 38: 110-114 (1995)) and site directed mutagenesis PCR (Madsen et al, Immunogenetics 40: 37-44 (1994)).
Mutations in the protein can also be detected by a variety of other means including direct DNA sequencing, Southern blot hybridisation or by using peptide specific antibodies, i.e. directed against the mutated protein.
In a further aspect, the present invention provides a kit for use in any of the methods of the invention which comprises one or more pairs of suitable primers for use in PCR amplification of the exon 1 region of the MBP gene.
Optionally, the kits of the invention further comprise one or more reagents/materials for use in establishing the MBP genotype of a subject. Examples of suitable reagents for establishing the genotype include primers for use in SSO dot-blot hybridization.
In yet further aspects, the present invention provides:
(a) the use of MBP in the preparation of a medicament for the prevention or treatment of a chronic viral infection;
(b) the use of MBP in the preparation of a medicament for the prevention or treatment of chronic fatigue syndrome;
(c) a method for the prevention or treatment of chronic fatigue syndrome which comprises administering to a subject an effective amount of MBP;
(d) a method for the prevention of a chronic viral infection which comprises administering to a subject an effective amount of MBP;
(e) the use of MBP in the preparation of a medicament for the treatment of HBV infection;
(f) the use of MBP in the preparation of a medicament for the prevention of acute HBV infection;
(g) the use of MBP in the preparation of a medicament to prevent development of chronic HBV infection;
(h) a method for the treatment of HBV infection which comprises administering to a subject an effective amount of MBP;
(i) a method for the prevention of acute HBV infection which comprises administering to a subject an effective amount of MBP; and
(j) a method for preventing development of chronic HBV infection which comprises administering to a subject an effective amount of MBP.
(k) a method for the treatment of recurrent infection in a child which comprises administering to the child an effective amount of MBP.
Given that a clear link has been established between the codon 52, 54 and 57 mutations of the MBP gene and the appearance of certain conditions, the possibility exists that patients could be treated by one or other forms of xe2x80x9cgene therapyxe2x80x9d. In this way a patients defective MBP gene could be corrected/repaired/replaced resulting in the provision of normal MBP in the patient.
In an additional aspect the present invention therefore provides:
(I) the use of DNA encoding the normal form of the MBP gene in the manufacture of a medicament for use in the prevention or treatment of:
(i) a chronic viral infection, eg chronic HBV infection;
(ii) chronic fatigue syndrome;
(iii) depressive disease;
(iv) irritable bowel syndrome;
(v) gulf war syndrome; and/or
(vi) recurrent infection, particularly bacterial or fungal infection.
In the methods of the present invention a suitable pair of primers for performing PCR amplification of the DNA sample are:
5xe2x80x2 primer: 5xe2x80x2-GCACCCAGATTGTAGGACAGAG-3xe2x80x2 SEQ ID NO.:1; and
3xe2x80x2 primer: 5xe2x80x2-CAGGCAGTTTCCTCTGGAAGG-3xe2x80x2 SEQ ID NO.:2
and these primers form yet a further aspect of the present invention.
In another embodiment determination of a subject""s genotype is conveniently carried out using sequence-specific oligonucleotide (SSO) dot-blot hybridization and suitable primers are as follows:
codon 52 wild type: 5xe2x80x2-GATGGGCGTGATG-3xe2x80x2 SEQ ID NO.:3;
codon 52 mutant: 5xe2x80x2-GATGGGTGTGATG-3xe2x80x2 SEQ ID NO.:4;
codon 54 wild type: 5xe2x80x2-GTGATGGCACCAA-3xe2x80x2 SEQ ID NO.:5;
codon 54 mutant: 5xe2x80x2-GTGATGACACCAA-3xe2x80x2 SEQ ID NO.:6;
codon 57 wild type: 5xe2x80x2-ACCAAGGGAGAAAAG-3xe2x80x2 SEQ ID NO.:7; and
codon 57 mutant: 5xe2x80x2-ACCAAGGAAGAAAAG-3xe2x80x2 SEQ ID NO.:8
and these primers form another aspect of the present invention.
Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis. 
The invention will now be described with reference to the following examples, which should not be construed as in any way limited the invention.